Flow Cytometry Antibody

1. Project Specifications

For each case, our team explores with the client the challenges of the project and together, they define the specifications.

• The target of interest : membrane protein, intracellular protein, peptide, small molecule, post-translational modifications, etc.
• Applications : Cytometry/FACS, functional tests, diagnostic or therapeutic application, etc.
• Specifications : Available material (protein, cell line, etc.), specificity, deadlines, etc.

Taking into account these multiple parameters, BIOTEM proposes tailor-made strategies to meet the client's needs and guarantee the success of the project.

2. Immunization

To meet each precise demand and client’s specifications, BIOTEM relies on a wide range of proven immunization strategies to obtain high affinity and specificity antibodies. Specific methods are developed to ensure that the appropriate immune response is achieved. Serum will be tested by Flow Cytometry and ELISA. The client will also have the possibility to test the sera in his/her application. At the end of this phase BIOTEM will be able to propose a contract with guaranteed results.

• Protein Immunization : From the production of the recombinant protein (transient transfection in CHO) to its preparation for immunization
• Peptide Immunization : BIOTEM has the experience and know-how to optimally integrate all the parameters and constraints of the design of immunogenic peptides. Immunization with peptides is a relevant strategy for the generation of antibodies directed against selected epitopes. This approach also reduces the need for sophisticated protein preparations for immunization.
• Genetic immunization: Genetic immunization is one of the strategies of choice for the development of monoclonal antibodies directed against membrane proteins, for example. This technique involves the use of messenger RNA (mRNA) or complementary DNA (cDNA) encoding target proteins to stimulate the production of specific antibodies against these proteins. This approach can promote the production of high-affinity, high-specificity antibodies, which are essential for effective targeting of membrane proteins.
• R.A.D^® Protocol (Exclusive) : Rapid Antibody Development allows the fast development of hybridomas in less than a month (immunization, fusion and screening included) and offers the possibility to work on highly conserved targets.
• S.S.I. Protocol (Exclusive) : Small Size Immunogen, optimized for small targets (< 30 kDa).
• Subtractive immunization : These techniques are particularly useful for the generation of antibodies against epitopes that are poorly immunogenic and/or sharing a high degree of homology
• Other - Small molecules (haptens), Genetic, cellular or in vitro immunization : Suitable solutions when immunogens are difficult to obtain or in the case of toxic targets.

3. Lymphocyte Hybridization & Hybridoma Screening

Depending on the Phase I results, the client will decide whether to launch subsequent phase or not (Go / No-Go). With its expertise, BIOTEM offers optimized screening methods to identify the best hybridomas (oligoclonal stage).

• Fusion/ Lymphocyte Hybridization: Fusion of the lymphocytes with myeloma cells to produce hybridomas (immortalization)
• Antibody Screening: A crucial step in the project to isolate reactive and specific clones. The methods used are adapted for each project according to the client's specifications (standard, competitive, multiple screening, etc.). Several screening rounds are generally carried out. BIOTEM offers Flow Cytometry screening.
• Confirmations: The positive clones from the screening are then retested by Flow Cytometry and ELISA if necessary. At this stage the client will have the possibility to test the positive clones
• Cell Bank: After the tests performed by the client, the selected hybridomas are secured.

4. Hybridoma Cloning

The selected hybridomas are then stabilized by limiting dilution. This step is necessary to obtain monoclonal lines. Functionality will be checked by Flow Cytometry before a cell bank (cryotubes) is validated.

The client will have the option to receive intermediate supernatants to test in his/her application.

The cryogenic vials can be transferred to the client or stored in secure containers at BIOTEM for future productions. (Learn more).

5. Antibody Production & Purification

BIOTEM offers different solutions for the production and purification of antibodies, from pilot batch to large-scale. Productions are systematically offered in Low Bovine IgG conditions and can be Low Endotoxin (< 10 EU/mg; even < 1 EU/mg) on request.

• In vitro Production : Flask, HYPERFlask®, CELLine, Bioreactors, etc.
• Purifications : Affinity chromatography, precipitation, size exclusion, etc. Learn more
• Evaluation by Flow Cytometry

Additional services such as antibody characterization (sequencing, affinity, etc.) or modification (coupling, fragmentation, etc.) are also available.